BackgroundMyocardial infarction is a life-threatening event, and timely intervention is essential to improve patient outcomes and mortality. Previous studies have shown that the time to thrombolysis should be less than 30 minutes of the patient’s arrival at the emergency room. Pain-to-needle time is a time from onset of chest pain to the initiation of thrombolysis, and door-to-needle time is a time between arrival to the emergency room to initiation of thrombolytic treatment. Ideally, the target for door-to-needle time should be less than 30 minutes; however, it is unclear if the door-to-needle time has a significant impact on patients presenting later than three hours from the onset of pain. As many of the previous studies were conducted in first-world countries, with established emergency medical services (EMS) systems and pre-hospital ST-elevation myocardial infarction (STEMI) triages and protocols, the data is not completely generalizable to developing countries. We, therefore, looked for the impact of the shorter and longer door-to-needle times on patient outcomes who presented to the emergency room (ER) with delayed pain-to-needle times (more than three hours of pain onset). ObjectiveTo determine the impact of delayed pain-to-needle time (PNT) with variable door-to-needle time (DNT) on in-hospital complications  in patients with ST-elevation myocardial infarction (STEMI) who underwent thrombolysis.Methods and resultsA total of 300 STEMI patients who underwent thrombolysis within 12 hours of symptoms onset were included, which were divided into two groups based on PNT. These groups were further divided into subgroups based on DNT. The primary outcome was in-hospital complications between the two groups and between subgroups within each group. The pain-to-needle time was ≤3 hours in 73 (24.3%) patients and >3 hours in 227 (75.7%) patients. In-hospital complications were higher in group II with PNT >3 hours (p <0.05). On subgroup analysis, in-hospital complications were higher with longer door-to-needle time in group II (p<0.05); however, there was no difference in complications among group I.ConclusionOur study is consistent with the fact that shorter door-to-needle time, even in patients with delayed PNT (>3 hours), has a significant impact on in-hospital complications with no difference in mortality. Restoration of blood flow to the culprit artery is the main principle for the treatment of acute ST-segment elevation myocardial infarction (STEMI). This has been proven to decrease the size of infarction and improve overall survival; however, according to Global Registry of Acute Coronary Events (GRACE) data, up to 40% of patients presenting acutely with STEMI fail to receive appropriate reperfusion therapy .Timely reperfusion of ischemic myocardium is the cornerstone in the management of STEMI. The latest American & European guidelines suggested a “door-to-needle time” (DNT), which is from arrival to the hospital till initiation of thrombolytic injection should be less than 30 minutes, is taken as the current gold standard and quality metric for timely reperfusion via pharmacological thrombolysis .The “pain-to-needle time’’ (PNT), which is the time taken from the onset of chest pain till the administration of the thrombolytic injection, represents the total time the artery was occluded, and the myocardium was deprived of blood flow. Mortality reduction can be up to 50% if thrombolysis is started within 180 minutes, i.e., three hours of symptom onset . This thAs per GRACE data, almost up to 40% of patients present late and end up missing this time window for thrombolytic therapy . All theWe performed a single-center retrospective cohort study by extracting information from institutional data of patients hospitalised for STEMI between July 2015 and December 2015. A total of 300 adult patients who underwent thrombolysis for the first presentation of ST-elevation myocardial infarction within 12 hours of symptoms onset were included. The patients with a previous history of thrombolysis, prior myocardial infarction (MI), heart failure with Killip class IV, and prior LV dysfunction documented on the previous echocardiogram were excluded.The data recorded comprised of patient's demographics, Electrocardiogram (ECG) changes, pain-to-needle time (PNT), door-to-needle time (DNT), and in-hospital complications  and echocardiographic parameters. Outcomes were assessed based on chart review and documentation on hospital discharge. Permission from the institutional review board (IRB) was obtained before the commencement of the study. The patients were divided into two groups based on early and delayed PNT . Each group was further sub-divided based on shorter (≤30 min) vs. longer (>30min) DNT  between 35%-50%, moderate if EF between 25%-35%, and severe if EF was less than 25%. IBM Corp. Released 2015. IBM SPSS Statistics for Windows, Version 23.0. Armonk, NY: IBM Corp. was used for data entry and analysis. Mean and standard deviation were calculated for age, pain-to-needle, and door-to-needle times. Frequency and percentages were calculated for gender, diabetes, hypertension, smoker, delayed DNT, delayed PNT, and in-hospital complications. The Chi-square test was used for qualitative variables. P-value ≤ 0.05 was taken as significant.Table There were 266 male and 34 female patients included in the study. The mean age of study subjects was 54.99±7.74 years, with the mean duration of chest pain being 196.15±87.85 minutes. Out of all patients, 42.0% of the patients were diabetic, 48.0% were hypertensive, 59.3% were smokers, and 17.7% were obese.The pain-to-needle time of 73 patients was ≤3 hours , while it was 3-12 hours in 227 patients . Group I and II were further divided into shorter (≤30 minutes) and longer DNT (>30 minutes) times. In group I, shorter DNT was observed in 41 patients, while longer DNT was observed in 32 patients. In group II, shorter DNT was observed in 142 patients, while longer DNT was observed in 85 patients. See Figure There was a significant association of pain-to-needle time observed with age p<0.0001), diabetes mellitus (p<0.0001), hypertension (p<0.0001), smoking, obesity (p=0.012) , post-infarct angina was observed in 2.7% in group I vs. 22.9% in group II with a P-value of 0.001. Heart failure with Killip II was observed in 8.2% vs. 11.45%, while heart failure with Killip III was observed in 0% vs. 7.9% (P=0.001). Mild LV dysfunction was observed in 12.3% in group I vs. 9.6% in group II, moderate LV dysfunction in 5.4% vs. 57%, severe LV dysfunction in 2.7% vs. 5.7% . Death was observed in 0% in group I while 2.2% in group II with a P-value of 0.340. Therefore, there was a significant difference in post-infarct angina, heart failure, and LV dysfunction when thrombolytics were given within three hours of pain, with no difference in death between these groups.  is managed on the guiding principle of restoring blood supply to the ischemic myocardium, which decreases morbidity and mortality, and primary percutaneous coronary intervention (PPCI) is the treatment of choice in this setting. As evidenced by multiple large trials, up to a third of these patients with AMI don’t get these timely lifesaving interventions, and up to two-thirds of these patients are those that seek help but initially present to setups that don’t have the facility of performing PPCI ,8. In thThe European Society of Cardiology emphasizes timely reperfusion such that they came up with the famous quote “time is muscle”. Therefore, the survival benefit is governed by an entire chain of events starting from early recognition of symptoms to the prompt delivery of emergency services (EMS) and prompt transfer to a PPCI capable hospital ,11. MultPre-hospital thrombolysis can serve as a viable alternative when PPCI isn’t possible within 90 minutes. A caveat here is the availability of advanced cardiac life support (ACLS)-equipped ambulances and trained staff for administering these therapies and monitoring their effects. Abba et al. showed that thrombolytic therapy was administered to the majority of patients presenting with acute MI within two hours of their hospital arrival . A studyPrompt thrombolysis and CCU care within one-and-a-half hours of symptom onset translate into smaller infarcts sizes, more preservation of left ventricular function, and lower 21-day mortality . The resOur observations are also in line with older studies mentioned above that there is no difference in mortality outcomes in early arrivals, i.e., within three hours. However, prompt administration of thrombolysis within 30 minutes of arrival in late arrivals, i.e., more than three hours, did show a significant trend towards less post-infarct angina, less LV dysfunction, and less heart failure. We, therefore, conclude that DNT should take precedence in improving outcomes on in-hospital complications in late arrivals. However, we did not find any significant difference in mortality between either of the groups.Study limitationsThis is a retrospective study and information based solely on chart documentation. Our study is limited by its single location in an urban environment. We only included those patients who mentioned chest pain with ST-elevation MI, and possibly we could have missed patients with acute coronary syndrome (ACS) equivalent symptoms such as shortness of breath, weakness, and neck/shoulder/back pain. Enzymatic infarct size was not available in all patients, with a potential underestimation of the impact of early treatment on myocardial salvage.The results indicate that even with the delay in presentation, rapid administration of thrombolytic therapy improves in-hospital outcomes. Any delay in door-to-needle or pain-to-needle time in STEMI patients was associated with increased in-hospital complications. Thus, reducing this time delay to the greatest extent should be made possible for such patients. A collaborative approach between the Emergency Department and the cardiology team and better prehospital triage and transfer services can improve delays and potentially improve outcomes. In a resource-limited country, all efforts should be made to decrease these delays to provide better patient care and increase chances of survival.


Purpose: To investigate the effectiveness and safety of 577-nm subthreshold micropulse laser (SML) on acute central serous chorioretinopathy (CSC).Methods: One hundred and ten patients with acute CSC were randomized to receive SML or 577-nm conventional laser (CL) treatment. Optical coherence tomography and best-corrected visual acuity (BCVA) were performed before and after treatment.Results: At 3 months, the complete resolution of subretinal fluid (SRF) in 577-nm SML group (72.7%) was lower than that in CL group (89.1%) , but it was 85.5 vs. 92.7% at 6 months . The mean LogMAR BCVA significantly improved, and the mean central foveal thickness (CFT) significantly decreased in the SML group and CL group  at 6 months. But there was no statistical difference between the two groups . In the SML group, obvious retinal pigment epithelium (RPE) damage was shown only in 3.64% at 1 month but 92.7% in the CL group (P < 0.001).Conclusions: Although 577-nm SML has a lower complete absorption of SRF compared with 577-nm CL for acute CSC at 3 months, it is similarly effective as 577-nm CL on improving retinal anatomy and function at 6 months. Importantly, 577-nm SML causes less damage to the retina. Central serous chorioretinopathy (CSC) is a common macular condition affected mainly in middle-aged patients. It is characterized by a serous neuroepithelium detachment with or without retinal pigment epithelium (RPE) detachment . The acuPhotodynamic therapy (PDT) and conventional laser (CL) are the mainly proved methods. Although PDT is effective for CSC, it causes ischemia and atrophy of the choroid . BesidesA 577-nm subthreshold micropulse laser (SML) is a reliable and cost-effective treatment. Furthermore, the 577-nm wavelength is yellow light and is outside the absorption spectrum of retinal xanthophylls, which potentially facilitates treatment close to the fovea . RecentlThis was a single-center, randomized, controlled trial of 577-nm SML vs. 577-nm CL to treat acute CSC, which was registered on ClinicalTrials.gov (identifier: NCT02784665). The study was carried out at Zhongshan Ophthalmic Center (ZOC), Sun Yat-sen University in China, from June 2016 to March 2018. Patients were randomized at a ratio of 1:1 into the 577-nm SML group and 577-nm CL group by block randomization, with a block size of 10. The randomization sequence was generated using a computerized randomization stable. All subjects were masked to the treatment allocation groups and gave informed consent before treatment. The study was adhered to the tenets of the Declaration of Helsinki and approved by the Ethics Committee of ZOC.Acute CSC was defined as persistent SRF for <6 months. The following inclusion criteria were fulfilled: patients between 18 and 55 years of age, first episode, visual symptoms related to CSC for at least 4 weeks, active leakage away from foveal (more than 300 μm) on fundus fluorescein angiography (FFA), abnormal dilated choroidal vasculature on indocyanine green angiography (ICGA), and SRF involving the fovea on spectral-domain optical coherence tomography (SD-OCT). The exclusion criteria were as follows: patients who underwent previous treatment, including PDT, focal laser photocoagulation, intravitreal injection treatment with anti-vascular endothelial growth factor (VEGF); with other fundus diseases such as polypoidal choroidal vasculopathy (PCV), choroidal neovascularization (CNV), other retinal vascular disorders and maculopathies; high myopia; patients receiving the treatment of exogenous corticosteroid systemically; pregnancy; inability to perform relative fundus examination.All patients received complete eye examinations at baseline and followed up at 1, 3, and 6 months after treatment. Best-corrected visual acuity was measured using the decimal chart and was converted to the logarithm of the minimum angle of resolution (LogMAR) for statistical analysis. Fundus angiography  was performed to determine the leakage spot and to exclude other maculopathies at baseline. SD-OCT and fundus autofluorescein  were performed at baseline and each visit. The central foveal thickness (CFT) was defined as the distance from the neurosensory retina's inner surface to the inner surface of the choroid at the fovea measured by OCT. RPE change was assessed using the FFA at the 1-month visit after laser treatment. RPE was categorized into: no RPE damage (no changes at the treatment area), mild RPE damage , and obvious RPE damage (presence of clear laser spots).CL group was treated with a 577-nm laser  using a continuous-wave model with a 100-μm spot diameter, a 0.1-s duration, and 80–120 mW power. A slight gray spot was the endpoint of CL. The micropulse mode of the 577-nm laser was used for the SML group. The micropulse treatment parameters were standardized for all patients, with 100 μm spot size, 200 ms duration, and a 5% duty cycle. The titration was individualized and operated in the normal retina outside the vascular arcades. The titration power was started at 600 mW with a monospot micropulse model and increased gradually until a just visible minimal graying reaction was seen as the threshold burn. Then the laser power was reduced to 50% as the treatment power of SMPL. Titration power ranged from 800 to 1,200 mW. Hence, the treatment power was between 400 and 600 mW. The micropulse laser in a dense pattern overlaid the leakage points, and the number of micropulse spots was <50 in one session. Treatment was performed using the Mainster contact lens .If the SRF involved in the macular was still present at the 3-month follow-up, the same intervention was repeated. And the SRF of all patients was assessed again as the second outcome at 6-month follow-up.The primary outcome was the complete absorption rate of the SRF based on the OCT images at 3 months. The secondary outcomes included changes in the BCVA and CFT at every visit and the complete absorption rate of the SRF at the final endpoint (at 6 months). At the same time, we evaluated the damage of RPE based on FAF imaging at 1 month.The sample size was designed to enroll 110 patients based on an estimated rate of complete SRF absorption rate at 3-month follow-up of 75% for the 577-nm SML group and 95% for the 577-nm CL group, with 80% power at a 2-sided α of 0.05 to detect a difference between two groups, estimating 20% loss to follow-up. The sample size was calculated with PASS 11 .t-test for continuous variables with normal distribution, the Wilcoxon rank-sum test for continuous variables with non-normal distribution, the chi-square test, or Fisher exact test for categorical variables. The complete absorption of SRF at a 3-month follow-up, the primary outcome, with a relative risk (RR) and 95% confidence intervals (CIs), was calculated and compared.According to the intention-to-treat analysis principles that all randomized participants were included, we used the last observation carried forward (LOCF) method to impute the missing values at 3 months and the final endpoint. Results were presented as the mean (SD) or median (IQR) for the continuous variables and frequency for categorical variables. Baseline data comparisons between the two groups were performed by a 2-tailed p < 0.20 level in a simple regression model were added to the multiple regression model. All statistical analysis was performed using SAS statistical software, version 9.4 . A p-value with <0.05 was considered statistically significant.For the potential prognostic factors at baseline, variables with Among 118 eligible patients confirmed as acute CSC with <6-month duration, three (2.54%) did not fit the inclusion criteria, and five (4.24%) declined participation. The remaining 110 patients (93.2%) were enrolled, of which 55 patients (50.0%) were randomly allocated to the 577-nm SML group and 55 patients (50.0%) to the 577-nm CL group. At 3 months, the primary outcome was assessed, and five patients (4.54%) were lost to follow-up: three patients were too busy to follow up, one patient moved to another hospital, and one patient transferred to another place. At 6 months, the secondary outcome was assessed, and eight patients (7.27%) were lost to follow-up: six patients were too busy to follow up, one patient moved to another hospital, and one patient transferred to another place . All misP = 0.113) and mean CFT .The baseline demographic and clinical characteristics were balanced between the two groups and are summarized in P = 0.029).For the primary outcome at 3-month follow-up, the patients with acute CSC in the SML group had a lower complete resolution rate of SRF [40/55 (72.7%)] compared with patients in the CL group [49/55 (89.1%)], with the significant difference  and baseline BCVA (LogMAR) . Other baseline characteristics, including age, sex, duration, and baseline CFT, were not significantly associated with the complete resolution of SRF .The patients who were still present SRF involved in the macula at a the 3-month follow-up received the same intervention as the baseline: 14 patients in the SML group and five patients in the CL group. And the SRF resolution of all patients was assessed again as the other outcome at the final endpoint (6-month follow-up). The complete resolution of SRF reached 85.5% (47/55) in the SML group and 92.7% (51/55) in the CL group, but there was no significant difference between the two groups . In the 577-nm CL group, the visual acuity (LogMAR) was markedly improved from 0.39 ± 0.22 at baseline to 0.08 ± 0.16 at 1 month, 0.01 ± 0.11 at 3 months, and 0.00 ± 0.09 at 6 months . The change of BCVA (LogMAR) in the SML group was lower than that in the CL group with an unadjusted difference . However, there was no statistical difference concerning the change of BCVA (LogMAR) at the final endpoint between the two treatment groups   had a statistically significant improvement from baseline to the endpoint in the two groups. The mean BCVA in the 577-nm SML group was 0.11 ± 0.17 at 1 month, 0.03 ± 0.12 at 3 months, and 0.00 ± 0.10 at 6 months, respectively, all of which was significantly improved compared with the visual acuity of the baseline (0.32 ± 0.21)  .p < 0.001). The mean CFT was 482 ± 155 μm at baseline in the 577-nm CL group and decreased remarkably to 226 ± 77.1 μm at 1 month, 210 ± 36.6 μm at 3 months, and 214 ± 35.6 μm at 6 months . However, there was no statistical difference for the change of CFT at the 3-month visit  and at the 6-month visit  between the two treatment groups . Besides, during the 6-month follow-up, choroidal neovascularization was not seen on OCT imaging in all patients.Based on the FAF images, RPE damage was evaluated at a the 1-month follow-up. In the 577-nm SML group, 69.1% (38/55) patients showed no RPE damage, 27.3% (15/55) had mild RPE damage, and only 3.64% (2/55) showed obvious RPE damage. The corresponding data in the 577-nm CL group were 0.00% (0/55), 7.27% (4/55), and 92.7% (51/55), respectively . The patTo the best of our knowledge, this study is the first prospective randomized controlled trial on the comparison of 577-nm SML with 577-nm CL for acute CSC. Our study showed that the SRF of patients with acute CSC in the 577-nm SML group had a lower complete absorption rate in the short-term (at 3 months), compared with that in the CL group. But after retreatment, 577-nm SML can reach a similar effect on improving the functional and anatomical outcomes of eyes with acute CSC. Importantly, 577-nm SML scarcely damaged RPE compared with CL.p < 0.001). Accordingly, visual acuity improved significantly from 0.39 ± 0.22 LogMAR at baseline to 0.00 ± 0.09 LogMAR (p < 0.001) after treatment. But evident laser scar (RPE damage) was seen in 51 (92.7%) patients, significantly higher than that of 577-nm SML.The conventional laser has been applied to retinal disease for many years, and it works by thermal energy to the RPE . NeverthMinimizing the adverse reactions of CL treatment, new modalities have emerged over the years . SML, deRecent retrospective studies have demonstrated that 577-nm SMPL could achieve the equivalent effect as conventional laser but without RPE damage . It has There is a challenge for SML treatment, especially in power selection. Power titration is an essential step in SML treatment, and it is usually outside the vascular arcades. The method of titration was reported in several clinical studies , 15, 25.There were several limitations to our study. First, we cannot assess and compare the long-term effect of 577-nm SML with 577-nm CL due to a half-year follow-up. Additionally, an observation group was absent not to eliminate the spontaneous resolution of SRF after the SML. It was reported that acute CSC was resolved spontaneously for 3–6 months and with 57% complete absorption of SRF in previous studies , 3, 27. In conclusion, our results revealed that 577-nm SML had a lower complete absorption of SRF for acute CSC than 577-nm CL at the 3-month follow-up. But it reached a similar effect on the improvement of anatomy and retinal function at 6 months between the two groups. The findings suggest that SML works gently compared to the CL. Furthermore, 577-nm SML treatment was safer because it had no apparent damage to RPE and could be used repeatedly, even be applied to the macular disease close to the fovea. Therefore, SML is a potential treatment option for patients with acute CSC by accelerating the absorption of SRF and improving the visual function without evident visible retinal damage.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of Zhongshan Ophthalmic Center. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.CJ: conceptualization. LZ and KL: methodology and writing—original draft preparation. CH: validation. FX: formal analysis. YG: investigation. LZ and LJ: data curation. ZZ and CJ: writing—review and editing. LLi: supervision. LLu: project administration. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The braces of today are constructed to correct the frontal plane deformity of idiopathic adolescent scoliosis (AIS). The Spinaposture brace©  is a soft-fabric brace for AIS and is designed to enhance rotational axial stability by inducing a sagittal plane kyphotic correction. This prospective observational study evaluated the brace in fifteen patients with AIS. The initial average CA was 16.8° (SD: 2.8). They were followed prospectively every 3 to 6 months during their brace usage until skeletal maturity of 25 months and at long-term follow-up of 44 months. In- and out-of-brace radiographs were performed in six subjects at inclusion. This resulted in an immediate in-brace correction of 25.3 percent in CA (14.3°→10.8°) and induced a kyphotic effect of 14.9 percent (40.8°→47.9°). The average in-brace improvement at first follow-up was 4.5° in CA, and the CA at skeletal maturity was 11° (SD: 7.4°) and long-term 12.0° (SD: 6.8°). In conclusion, the Spinaposture brace© had an immediate in-brace deformity correction and a thoracic kyphotic effect. At skeletal maturity, the deformities improved more than expected when compared to that of the natural history/observation and similar to that of other soft braces. No long-term deformity progression was seen. To substantiate these findings, stronger designed studies with additional subjects are needed. Idiopathic adolescent scoliosis (AIS) is a structural spinal disorder that sometimes requires the intervention of either bracing or surgery due to the magnitude of or progression in the spinal deformity ,2. The bThe brace is intended to augment axial stability by inducing a sagittal plane thoracic kyphotic effect and, if needed, a derotation in the spine depending on the individual curve pattern. The brace is intentionally built to induce minor to no direct frontal plane correction, and to realign or restore the thoracic kyphosis by inducing an inherently proprioceptive ‘hyper-kyphotic strive’. The strive is induced utilizing the principle of autocorrection derived from physiotherapeutic practice . SpecifiThe subjects were patients referred with AIS from our hospital service area and included as a convenience sample. At the first visit, the including doctor diagnosed them with AIS, and if having a thoracic or thoracolumbar AIS with a CA between 15° and 25°, they were included after informed consent. The subjects and caregivers were specifically told that longitudinal radiological follow-up without bracing was the ‘gold standard’ treatment. Exclusion criteria were subjects with primary lumbar scoliosis, previous brace treatment or inability to cope with bracing in general, or if MRI-verified intraspinal pathology was present. If the subjects had a correction of approximately one-fourth in the frontal plane deformity after three months, when using the brace, this was continued. If the correction was less than one-fourth then the ‘hard shield’ was introduced. If the subjects had a correction of approximately one-fourth in the frontal plane deformity after another three months, when using the brace, then this was continued. Otherwise, the subjects were excluded. The ‘body stocking’ part of the brace was worn in the daytime (<16 h and defined as part-time) to counteract the effect of gravity on the spine and was not worn at night ,22. We aAt study initiation, the subjects were asked to volunteer to have performed initial in-brace radiographs . The subjects were followed prospectively with anteroposterior out-of-brace radiographs every six months in general. However, the initial radiographs were taken every three months as a precaution since we had no previous experience using the brace . The subThe brace was worn until skeletal maturity. Skeletal maturity was determined by having at least Risser 4, duration of menstruation of 2 years for girls, and a skeletal hand age of 14 years and 16 years for girls and boys (Sanders stage 8), respectively. The follow-up was continued for an additional three to six months with a final radiographical and clinical evaluation. The subjects were recalled for one long-term radiographic follow-up. The primary outcome was Cobb’s angle for the primary curves at skeletal maturity. A change of five degrees or more was considered either a progression or regression ,25. If tSecondary parameters were radiographic and evaluated as categorical variables of changed or unchanged. The parameters were the descriptive categorization of scoliosis according to the Moe-Kettleson classification , changes® HealthCare, Mortsel, Belgium) on a three-megapixel viewing station. For statistical analyses, we performed an inter-class correlation for inter-observer variability between the evaluators. The chi-square cross-tabulation test and the Spearman correlation test were performed for differences between subjects and drop-outs. All tests were performed using IBM SPSS Statistics, Version 25 . A post hoc power analysis was performed using GPower .The evaluators were two doctors of pediatric orthopaedic and spinal surgery with 26 and 20 years of experience, respectively. All measurements were performed separately and blinded using the Synedra View Personal 19  and the PACS system . Informed consent was obtained from all involved subjects. This research received no external funding.Twenty-two patients were screened. Seven patients were excluded from the study at first evaluation. Two subjects had CA larger than 25° and previous brace treatment, three subjects were not included due to syrinx or anisomelia, and two subjects received the brace but never used it. Fifteen subjects were included with no prior treatment, except for observation. Five subjects dropped out. Two subjects, after joining a formal Schroth therapy, acquired the Chêneau Gensigen brace by their own accord. This was encouraged by their private physiotherapists. One subject had back pain without improvement by bracing, one changed to Rolfing structural integration therapy, and one did not attend the follow-ups. All three subjects discontinued bracing. Two subjects did not attend the long-term follow-up. One subject was too busy with school work and we were unable to recall one. We considered them as drop-outs. Otherwise, there were no subjects lost in follow-up or withdrawals. For the ten subjects at skeletal maturity in the follow-up study, the average age at brace initiation was 12.26 (SD: 2.76) years. The clinical characteristics of the subjects and relevant medical history are summarized in Six subjects were examined for the in-brace correction with radiographs in the anteroposterior and lateral projections. The frontal plane correction and kyphotic effect were measured by comparing the CA measured at the same levels on the in- and out-of-bracing radiographs. The in-brace correction of the primary curve in the frontal plane was 25.3 percent (14.3° to 10.8°). The in-brace thoracic kyphosis in the sagittal plane increased 14.9 percent (40.8° to 47.9°). In the prospective follow-up, the average CA when starting bracing was 16.8° (SD: 2.8°). The average correction in CA at the first follow-up was 4.5° (SD: 3.2°). Initially, four subjects (4/15) improved in CA with more than 5 dg. and eleven (11/15) remained stable. The average bracing period was 21 months . The average CA at the end of bracing was 11.0° (SD: 7.2°). At end of bracing, six subjects (6/10) had straight spines with a CA less than ten degrees and four (4/10) remained stable. In accordance with Costa et al. (2021), the expected outcome for bracing at skeletal maturity was 6/11 and 9/15 for the Risser stage 0–1 and 0–2 at brace initiation, respectively . The achSecondary radiographic parameters in the follow-up study were evaluated according to if changed and are illustrated in The compliance of brace usage and events were evaluated by open questioning at every follow-up where the subjects and caregivers were encouraged to disclose non-compliance and discomfort when using the brace in an open-minded dialogue. Non-compliance and brace events were seen in periods of warm weather and the subjects often had subsequently deformity progression at the next follow-up. This motivated the subjects to a more rigorous brace usage motivated by the deterioration see . Two subWhen performing analysis for interobserver variation, the two doctors had an excellent inter-class correlation of 0.85 .When performing a posthoc power analysis, the power  for the first follow-up with 15 subjects was 0.98, at skeletal maturity 0.88 for ten subjects and long-term 0.79 for eight subjects. p < 0.001) and the duration of bracing (p < 0.01), respectively.When performing the Chi-square cross-tabulation, none of the parameters of gender, age, Risser stage, and initial CA between subjects and dropouts was statistically significant (z < 1.96). The Spearman correlation for dropout found a positive and negative association between initial CA (In this study, we examined a new brace for adolescent idiopathic scoliosis. When testing for immediate correction by in- and out-of-brace radiographs, the proprioceptive-mediated ‘strive’ of the brace induced a thoracic sagittal kyphotic effect. This led to an immediate thoracic frontal plane correction of the AIS deformity. This suggests that there can be an interrelationship between the frontal and sagittal plane spinal curves of the spine which is described as ‘coupled motions’ by Panjabi and White . To our When interpreting our results, we had the following deliberations. In the initial evaluations of the subjects, we performed in- and out-of-brace radiographs and closer follow-ups with three months intervals in the initial period of brace wearing as well. The in and out-of-brace radiographs were voluntary for the subjects since this induced additional radiation . We refrThe purpose of examining the brace was two-fold, namely to discover if the hypnotized frontal plane deformity would be correctable using a sagittal plane strive and to determine if the brace was effective at skeletal maturity and long-term. The hard braces used today are strenuous, causing pressure marks, pain/soreness, and general discomfort with low compliance to follow ,7,8. TheIn conclusion, this prospective observational study demonstrated an immediate in-brace correction of the frontal deformity of AIS in six subjects. In follow-up, there was an immediate correction of one-fourth of the deformity. At skeletal maturity, the deformities improved more than expected when compared to that of the natural history/observation and similar to that of other soft braces. No long-term deformity progression was seen.


The present study reports on the development of wood plastic composites (WPC) based on micronized argan shell (MAS) as a filler and high-density polyethylene obtained from sugarcane (Bio-HDPE), following the principles proposed by the circular economy in which the aim is to achieve zero waste by the introduction of residues of argan as a filler. The blends were prepared by extrusion and injection molding processes. In order to improve compatibility between the argan particles and the green polyolefin, different compatibilizers and additional filler were used, namely polyethylene grafted maleic anhydride (PE-g-MA 3 wt.-%), maleinized linseed oil (MLO 7.5 phr), halloysite nanotubes (HNTs 7.5 phr), and a combination of MLO and HNTs (3.75 phr each). The mechanical, morphological, thermal, thermomechanical, colorimetric, and wettability properties of each blend were analyzed. The results show that MAS acts as a reinforcing filler, increasing the stiffness of the Bio-HDPE, and that HNTs further increases this reinforcing effect. MLO and PE-g-MA, altogether with HNTs, improve the compatibility between MAS and Bio-HDPE, particularly due to bonds formed between oxygen-based groups present in each compound. Thermal stability was also improved provided by the addition of MAS and HNTs. All in all, reddish-like brown wood plastic composites with improved stiffness, good thermal stability, enhanced compatibility, and good wettability properties were obtained. In the last decades, an increasing concern about environmental issues related to the great use of petrochemical-derived plastics has been rising, as well as the necessity to reduce the carbon footprint associated with the production processes of those polymers. This trend is driving the industry toward the use of polymeric materials derived from natural resources, with the objective of moving away from petroleum. Moreover, this fact has propitiated the use of biodegradable polymers, some of which are capable of decomposing in composting conditions. At the same time, they exhibit very similar properties to those of their petrochemical counterparts . One of Among those materials, “Wood plastic composites” (WPC) are becoming increasingly popular. This technology implies the addition of natural organic fillers obtained from wastes into polymer matrices, with the objective of decreasing their processing cost. At the same time, those fillers enhance their environmental value and vary their mechanical, physical and chemical properties . InitialIn this sense, high density bio-polyethylene (Bio-HDPE) is one of the most interesting polymer matrices, as it combines its natural origin with the ease of processability of polyolefins. This polymer is produced by conventional polymerization of ethylene obtained from catalytic dehydration of bio-ethanol , which iSeveral studies have been carried out to improve the compatibilization between those fillers and polymeric matrices. On the one hand, some of the methods that have been applied to improve this compatibility include the modification of the surface of the filler particles from the waste, such as mercerization, benzoylation, acetylation, silanation, or graft copolymerization ,24. On t2[Si2O5(OH)4].nH2O), where n is 2 for hydrated HNTs, and 0 for dehydrated HNTs [In recent years, vegetable oils have generated a great deal of interest as compatibilizing additives. Modified vegetable oils have been used as plasticizers, stabilizers, crosslinkers, compatibilizers, and so on. The use of epoxidized vegetable oils (EVOS) should be remarked, such as epoxidized soybean oil (ESBO) ,32, epoxted HNTs . Frost eted HNTs  reportedted HNTs . Becauseted HNTs . Numerouted HNTs ,44,45,46ted HNTs , polyamited HNTs , epoxy rted HNTs , polylacted HNTs ,51, and Argania spinosa) is a tropical plant that belongs to the Sapotaceae family, whose fruit is used in morocco to prepare oil [The objective of this work is the reuse of micronized argan shell (MAS) as a reinforcing organic filler in a Bio-HDPE matrix derived from sugarcane, with the objective of developing a wood plastic composite with enhanced properties compared to the original matrix following the trends proposed by the circular economy, which seeks to reuse or recycling the waste and to reintroduce them into the industry . Argan  of 20 g/10 min, measured with a load of 2.16 kg and a temperature of 190 °C. Micronized argan shell (MAS) was supplied by MICRONIZADOS VEGETALES S.L. (Benamejí (Córdoba), Spain) company. Halloysite nanotubes were supplied by Sigma Aldrich  with CAS number 1332-58-7.Bio-HDPE, SHA7260 grade, was supplied in pellets form by FKuR Kunststoff GmbH  and manufactured by Braskem . This green polyethylene has a density of 0.955 g cmInterlocking is one of the most important mechanisms in polymer composites. This mechanism depends on the filler shape. −1 range.In relation to the additives, polyethylene-grafted maleic anhydride (PE-g-MA) with CAS Number 9006-26-2 and MFI values of 5 g/10 min (190 °C/2.16 kg), was obtained from Sigma-Aldrich S.A. . This PE-based copolymer was selected due to its functionality. The maleinized linseed oil—MLO, VEOMER LIN was supplied by Vandeputte . This modified vegetable oil is characterized by a viscosity of 10 dPa s at 20 °C and an acid value comprised in the 105–130 mg KOH gBio-HDPE, PE-g-MA, MAS y HNTs were initially dried at 40 °C for 48 h in a dehumidifying dryer MDEO  to remove any residual moisture prior to processing to avoid the possibility of hydrolysis due to the moisture. Then, the corresponding wt.% of each component see  were mixTo transform the pellets into standard samples, a Meteor 270/75 injector from Mateu & Solé  was used. The temperature profile in the injection molding unit was 135 °C (hopper), 140 °C, 150 °C, and 160 °C (injection nozzle). A clamping force of 75 tons was applied while the cavity filling and cooling times were set to 1 and 10 s, respectively. Standard samples for mechanical and thermal characterization with an average thickness of 4 mm were obtained.3, according to ISO 868:2003. The impact strength was also studied on injection-molded rectangular samples with dimensions of 80 × 10 × 4 mm3 in a Charpy pendulum (1-J) from Metrotec S.A.  on notched samples (0.25 mm radius V-notch), following the specifications of ISO 179-1:2010. All mechanical tests were performed at room temperature, and at least six samples of each material were tested and the corresponding values were averaged.Mechanical properties were obtained with different tests like tensile test, shore hardness, and Charpy impact test. Tensile properties of Bio-HDPE/PE-g-MA/MAS blends were obtained in a universal testing machine ELIB 50 from S.A.E. Ibertest  as recommended by ISO 527-1:2012 with dog bone samples 1B specification. A 5-kN load cell was used and the cross-head speed was set to 5 mm/min. Shore hardness was measured in a 676-D durometer from J. Bot Instruments , using the D-scale, on rectangular samples with dimensions 80 × 10 × 4 mmThe morphology of fractured samples from Charpy tests, obtained from the impact tests, were studied by field emission scanning electron microscopy (FESEM) in a ZEISS ULTRA 55 microscope from Oxford Instruments . Before placing the samples in the vacuum chamber, they were sputtered with a gold-palladium alloy in an EMITECH sputter coating SC7620 model from Quorum Technologies, Ltd. . The FESEM was operated at an acceleration voltage of 2 kV.The most relevant thermal transitions of Bio-HDPE/PE-g-MA/MAS blends were obtained by differential scanning calorimetry (DSC) in a Mettler-Toledo 821 calorimeter . Samples with an average weight of 6–7 mg, were subjected to a thermal program divided into three stages: a first heating from 25 °C to 160 °C followed by a cooling to 0 °C, and a second heating to 250 °C. Both heating and cooling rates were set to 10 °C/min. All tests were run in nitrogen atmosphere with a flow rate of 66 mL/min using standard sealed aluminum crucibles with a capacity of 40 μL.The thermal degradation of the Bio-HDPE/PE-g-MA/MAS blends was assessed by thermogravimetric analysis (TGA). TGA tests were performed in a LINSEIS TGA 1000 . Samples with a weight of 15–17 mg were placed in 70 µL alumina crucibles and subjected to a dynamic heating program from 40 °C to 700 °C at a heating rate of 10 °C/min in air atmosphere. The first derivative thermogravimetric (DTG) curves were also determined. All tests were carried out at least three times in order to obtain reliable results.3 were subjected to a dynamic temperature sweep from −150 °C to 120 °C at a constant heating rate of 2 °C/min. The selected frequency was 1 Hz and the maximum flexural deformation or cantilever deflection was set to 10 µm.In order to obtain the thermomechanical properties of the Bio-HDPE composites a dynamical mechanical thermal analysis (DMTA) was carried out in a DMA1 dynamic analyzer from Mettler-Toledo , working in single cantilever flexural conditions. Rectangular samples with dimensions 20 × 6 × 2.7 mmThe colorimetric modifications of the samples were measured with a Konica CM-3600d Colorflex-DIFF2, from Hunter Associates Laboratory, Inc.  was used. Color coordinates (L*a*b*) were measured according to the following criteria: L* = 0, darkness; L* = 100, lightness; a* represents the green (a* < 0) to red (a* > 0); b* stands for the blue (b* < 0) to yellow (b* > 0) coordinate.Hydrophilicity/ hydrophobicity of each blend was assessed by contact angle measurements through time were carried out with an EasyDrop Standard goniometer model FM140  which is equipped with a video capture kit and analysis software . Double distilled water was used as test liquid, a drop of it was put in each sample and contact angle measurements were taken at 0, 5, 10, 15, 20, and 30 min after the administration of the water. For color and wetting measurement, traction samples were used to facilitate the measures.3) from each blend were first weighted in a balance and then put inside a beaker filled with distilled water, all of them wrapped with tiny pieces of a metal grid so they could sink. After that, the weight of all samples was measured in intervals of several hours the first day, and then measured each week for 14 weeks in order to evaluate the amount of taken up water. In every measurement, the moisture in the surface of the samples was removed with tissue paper. During the immersion time, the temperature was controlled to 23 °C.The water absorption capacity of the Bio-HDPE/PE-g-MA/MAS blends was evaluated by the water uptake method. Impact specimens  were employed in this test and the measurement was performed at room temperature.In order to obtain the interactions between the different elements, a chemical analysis of the Bio-HDPE/PE-g-MA/MAS blends was carried out by attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. Spectra were recorded using a Bruker S.A Vector 22  coupled to a PIKE MIRacleTM single reflection diamond ATR accessory . Data were collected as the average of 10 scans between 4000 and 500 cmp ≤ 0.05) by one-way analysis of variance (ANOVA) following Tukey’s test. Software employed for this propose was the open source R software V4.0.3 (http://www.r-project.org (accessed on 12 March 2021))The significant differences among the samples were evaluated at 95% confidence level  could not be determined because the tensile test machine reached its maximum elongation without breaking the sample. Those values are indicative of certain stiffness and high ductility for Bio-HDPE, as it has been also reported by other authors [As it can be observed in  authors . Incorpo authors ,27. Howe authors  observed authors . On the  authors  when tre2, which is a ductile behavior indicator. Bio-HDPE/PE-g-MA/MAS blend shows a reduction of impact strength down to 1.4 kJ/m2, confirming the great stiffness of the blend, probably due to the quantity of MAS utilized [2, which means a significant difference between the mentioned samples. This effect is probably associated with an interaction between argan particles and HNTs. Moreover, it is possible that HNTs presence leads to a good dispersion of the argan particles, as well as HNTs themselves, boosting the reinforcing effect of the fillers [Regarding the impact strength results, neat Bio-HDPE presents a value of 2.7 kJ/mutilized . The pre fillers . As it w fillers . Impact With regard to Shore D hardness, a similar trend to that observed in Young modulus (E) can be observed. HNTs sample shows the highest hardness 60.6), giving evidence of the reinforcing effect of nanotubes and MAS. A very similar value is displayed by the ternary blend Bio-HDPE/PE-g-MA/MAS (59.2), reflecting the hardening effect of MAS, composites without MLO did not show significant differences between them. As expected, MLO samples exhibit the lowest hardness (53.2), due to the great ductility this component bestows upon the blend. Ferri et. al. reported this same effect in PLA/TPS blends with different proportions of MLO [.6, givinInternal morphology of these materials is directly related to their mechanical properties. Presence of HNTs in the blends can be observed in argan particles in m) and crystallinity (Xc) values of every Bio-HDPE blend. Regarding thermograms, in this case only melting temperature can be observed, because glass transition temperature is really low for Bio-HDPE (−100 °C) [C of 66.3%. Similar values were reported by Quiles-Carrillo et al. [m up to 133.1 °C. This effect was also observed by Erding et al. [m, but reduces, along with argan, crystallinity down to 39.8%. MLO improves compatibilization and, therefore, argan particle dispersion in the polymer matrix, thus contributing to reducing polymer–polymer interactions and favoring crystal formation [−100 °C) . In the o et al. . After to et al. . HNTs ing et al. . Regardiormation . However5%), while its maximum degradation rate temperature was (degT) 447 °C. Additionally, after a single-phase degradation, its residual mass at 700 °C was 0.3%. Montanes et. al. [Thermal stability has also been studied.  et. al.  observed et. al.  when the et. al. ,70. Thes et. al. , leading et. al. .According to residual mass results, all samples have more residual mass than neat Bio-HDPE. This is especially logic with HNTs, as they are mostly inorganic and possess a very high degradation temperature . In thisThermomechanical properties of blends were studied by DMTA technique. g [g change given by the addition of MAS with different compatibilizers is not so significant. In the case of Bio-HDPE/PE-g-MA/MAS and Bio-HDPE/PE-g-MA/MAS/HNTs samples, a reduction of about 3–4 °C is produced, while MLO reduces it even further (5 °C). It is logic that the 7.5 phr MLO sample possesses the lowest Tg due to the plasticizing effect previously mentioned. Regarding the combination of HNTs (3.75 phr) and MLO (3.75 phr), they seem to exert a synergetic effect that moves the glass transition peak +1 °C approximately. This can be associated with a good dispersion of HNTs into the polymer matrix, which immobilize Bio-HDPE chains to some extent. Only with the superposition of all the effects commented before, a significant difference could be observed, HNT/MLO blend reduced the Tg to the lowest temperature (−114.3 °C).g , which ig . A secong . The Tg Color, luminance, and transparency are essential issues to be considered in materials that try to imitate wood or WPC, in this case reddish-like, as those parameters determine how similar in appearance are these materials to wood. L*a*b* color coordinates were measured on injection-molded samples in order to analyze the color variations and luminance. Luminance (L*) is indicative of lightness. Neat Bio-HDPE showed high brightness as a consequence of its characteristic white color, in contrast with the brown-like color of the other samples, which present low luminance as a result of the addition of fillers and compatibilizers. Referring to color coordinate a*, it is indicative of the color change between green (negative) and red (positive). Particularly, neat Bio-HDPE presents a negative value but close to 0, −2.29 in this case, because of the proximity of the sample to white color. Similar value was reported by Rojas-Lema et al.  in neat Colors showed by the materials studied here make them perfect candidates for wood plastic composites , presentOne of the main disadvantages of green composites or any material based on hydrophilic fillers is their tendency to absorb water. With the objective of evaluating the behavior of Bio-HDPE/PE-g-MA/MAS blends against water, contact angle was measured at different times after applying a water drop over each sample surface. A great contact angle is indicative of a poor affinity toward water. From all these results it can be concluded that all fillers and compatibilizers used do not significantly vary hydrophilicity of Bio-HDPE at first, but they do increase water absorption in case of exposure for long periods of time. This could be corroborated by the statistical study where only some significant differences appeared at the initial time, but these were clearly accentuated over the time due to the different behaviors of the blends.Additionally, water absorption capabilities under long time exposure were studied for each material by means of water absorption test. According to these results, although hydrophilicity provided by argan particles (MAS) could prove a disadvantage in some ambits, it could certainly give applicability to these materials in some other fields. One of the applications could be flowerpot fabrication, so that when the plant is irrigated, water excess is absorbed by the pot material over time.−1. First, PE-g-MA spectrum is very similar to that of Bio-HDPE, as it is a polyethylene-based (PE) compound. Absorption bands at 2840 and 2910 cm−1 are associated with the stretching vibration for -CH-, -CH2-, or -CH3 [−1 corresponds to the deformation vibration of -CH2- or -CH3 [−1 is due to (CH2)n rock when n ≥ 4 [−1, which are related to the stretching vibration of C-O and C-OH in polysaccharide rings in cellulose [−1 can also be appreciated due to the symmetric glycosidic stretching of C-O-C bond in polysaccharide compounds of cellulose. Moreover, little absorption bands in the 1500 and 1130 cm−1 region can be observed, which indicate the presence of characteristic groups of cellulose, hemicellulose, and lignin. Some examples of those groups are the peak at 1227 cm−1, related to the stretching vibration C=O of the acetyl group in lignins [−1, which corresponds to the vibration of aromatic rings in lignin [−1, is ascribed to non-cellulosic compounds  [−1, which is related to stretching vibration of C-H bonds in CH and CH2 groups, present in cellulose and hemicellulose [−1, associated to the stretching of O-H bonds in carbohydrates (cellulose and hemicellulose) [−1 are due to the stretching vibration of Al2OH- (each OH is linked to two Al atoms) [−1 are ascribed to the O-H stretching of inner-surface hydroxyl groups and the out-of-phase vibration of the inner surface hydroxyl groups, respectively [−1 is attributed to bending vibrations due to absorbed water [−1 is due to apical Si-O bonds, while 1022 and 682 cm−1 bands are associated with the perpendicular stretching of Si-O-Si bonds [−1 are consequence of the O-H deformation of inner-surface hydroxyl groups and O-H deformation of inner hydroxyl groups, respectively [−1, and can be ascribed to the O-H translation vibrations of halloysite O-H units [−1, corresponding to =C-H stretching of double carbon-carbon bonds, and those at 2925 and 2850 cm−1 refer to the antisymmetric and symmetric stretching vibration C-H of saturated carbon-carbon bonds (C-C), respectively [−1, indicative of the C=O carbonyl stretching vibration of the ester and maleic anhydride, respectively; another band at 1158 cm−1 is due to the stretching vibration C-O-C, C-O, and C-C in ester groups; the peak at 720 cm−1 is normally related to the out-of-plane C-H stretching vibration of saturated C-C bonds [−1 is due to C-H bending vibration, while the absorption peaks at 1862 and 1784 cm−1 are attributed to anhydride groups.Chemical composition of the injection-molded samples was analyzed by means of Fourier transformed infrared spectroscopy (FTIR).  or -CH3 . The pea or -CH3 . On the en n ≥ 4 . In the ellulose ,88. A pe lignins ; and then lignin . The lowllulose) . Anotherellulose . A finalllulose) . Regardil atoms) . Bands aectively ,91,92,93ed water , the banSi bonds . Peaks aectively . Two lasectively . Other b-C bonds . Finally−1, and they are related to the stretching vibration, bending deformations, and rocking of methylene groups (CH2) [−1 are associated with wagging and symmetric deformations of CH2 and CH3 groups, respectively. The addition of MAS compatibilized with PE-g-MA makes a band in the range 1025–1091 cm−1 to appear, which is mainly attributed to the formation of PE-g-MA dimers or oligomers, identified with the stretching of C-C and C-O bonds [−1, relative to the presence of C-O and C-OH bonds in MAS particles cellulose. The presence of HNTs in the blend deforms and increases the intensity of the peak at 1030 cm−1, due to the perpendicular Si-O-Si stretching, as it has been previously mentioned in the individual spectra of HNTs. Additionally, in HNTs samples a weaker band at 900 cm−1 can be observed, ascribed to the deformation of O-H groups [−1 region, which have been previously identified in the individual analysis of HNTs, associated with the stretching vibration of inner-surface O-H groups in the nanotubes. Regarding the MLO presence in the blend, it produces changes found at 1690 cm−1 approximately, related to the C=O stretching in hydrolyzed anhydride groups in MLO [−1 can be ascribed to the absorption of water, catalyzed by MLO and argan particles, which are polar compounds with great amount of functional oxygen-based groups and great affinity toward water. HNTs and MLO blend shows the same characteristic peaks described for each individual sample, but with less absorption intensity, as their concentration reduces from 7.5 phr to 3.75 phr.ps (CH2) . These p-O bonds . Moreoves in MLO . This peThese results seem to reveal certain compatibilization between elements. This fact is denoted by a general increase in the intensity of peaks related to oxygen-based groups, indicating a possible bonding between anhydride groups in PE-g-MA and MLO with hydroxyl groups in MAS and HNTs, increasing the affinity reciprocally within the polymer matrix.The present work shows the incorporation of argan shell wastes in wood plastic composites with biobased polyethylene matrix and different compatibilizers. The incorporation of this type of loads can be effectively used as new reinforcement elements in order to create parts prepared by conventional industrial processes for thermoplastic materials, in special injection molding. In relation to the mechanical properties, the incorporation of MAS (30 wt.%) with PE-g-MA (3 wt.%) into the Bio-HDPE matrix increases the Young modulus up to 846 MPa. This can be directly related to a proper distribution of MAS particles in the polymer matrix, leading to a good adhesion of the filler with the polymer and obtaining a more rigid material. Addition of HNTs in the blend showed an additional enhancement of stiffness. In this context, Bio-HDPE/PE-g-MA/MAS/HNTs sample presented the highest Young modulus of all blends (1126 MPa). The incorporation of MLO in the green composites increased 100% of the elongation at break of the Bio-HDPE/PE-g-MA/MAS blend. From a morphological point of view, the incorporation of argan particles in the BioHDPE matrix, showed great cohesion, which implies good interaction between both elements. Furthermore, the addition of MLO verified the results obtained in the mechanical properties, showing a more ductile fracture and confirming the plasticizing effect of MLO. However, the incorporation of the lignocellulosic filler leads to a reduction in thermal properties and a decrease in the crystallinity of the compound. In relation to the thermomechanical properties, the incorporation of HNTs highly augmented the storage modulus, providing values of 3111 and 1898 MPa. This is indicative of an increase in the stiffness of the material, confirming the results commented in the mechanical properties section, associated with a high dispersion of HNTs in the matrix. In general, the incorporation of argan particles and additives provided the materials with reddish-brown colors that allow their use in the manufacturing of wood-based products. In relation to one of the main drawbacks of green composites, it can be seen how the incorporation of lignocellulosic particles greatly increases the water absorption of the composites, generating certain disadvantages. In addition, the incorporation of the MLO increases the water absorption capacity of the composites, due to the plasticization effect it exerts over the Bio-HDPE matrix.The results obtained in this work indicate that it is possible to obtain WPC with high renewable content with Bio-HDPE, lignocellulosic fillers and natural additives. This type of green composites can greatly favor the generation of circular economies focused on giving added value to agri-food waste from the Mediterranean basin, also favoring the creation of highly efficient polymers at very competitive costs. The materials obtained in this work have proved to possess excellent properties at a reduced cost in comparison with the neat Bio-HDPE. High ductile properties, relatively high stiffness and good thermal stability were reported, as well as a visual appearance very similar to that of reddish-color woods, which is essential in a wood plastic composite. The samples showed certain hydrophilicity over time, which can prove to be a disadvantage, although it is unusual for these materials to be immersed in water for long periods of time. However, if it were the case, they have some applications too, as it is the fabrication of plant pots. All in all, the affinity between argan particles and Bio-HDPE has been successfully increased in this study with the use of compatibilizers, and their properties vastly displayed and demonstrated. This work opens a new line of research regarding the development of new materials formed by polar fillers and non-polar polymeric matrices.


Irinotecan (CPT-11) is clinically known to cause severe diarrhea and gastrointestinal damage. Recently, we have reported that CPT-11-induced gastrointestinal damage is associated with the upregulation of intestinal P-glycoprotein (P-gp) expression and decreased absorption of its substrate, dabigatran etexilate (DABE), using a rat model. However, the P-gp activity or its contribution to the decreased absorption remains unclear. The aim of this study was to quantitatively evaluate how P-gp activity changes in rats with CPT-11-induced gastrointestinal damage, as assessed by the absorption of digoxin (DGX), a typical P-gp substrate.Male Sprague-Dawley rats were intravenously administered CPT-11 at a dose of 60 mg/kg/day for 4 days to induce gastrointestinal damage. Then, the rats were administered DGX orally (40 μg/kg), after some of them were orally administered clarithromycin , a P-gp inhibitor. DGX (30 μg/kg) was administered intravenously to determine the bioavailability (BA). The rats’ DGX plasma concentration profiles were determined using LC-MS/MS.max) and area under the plasma concentration-time curve (AUCpo) of DGX, which does not contradict to the DABE study. Although in the CPT-11-treated group the BA of DGX was significantly decreased to 40% of the control value, CAM did not affect the BA of DGX in the CPT-11-treated group.CPT-11 treatment decreased the maximum concentration (CIncreased P-gp expression in rats with CPT-11-induced gastrointestinal damage is not necessarily associated with increased P-gp activity or contribution to the drug absorption in vivo. The decreased DGX absorption observed in this study might be attributable to other factors, such as a reduction in the absorptive surface area of the gastrointestinal tract. Irinotecan CPT-11) is a topoisomerase-I inhibitor, which has been widely used as antineoplastic agent in the clinical setting since the late 1980s 1 is a to, 3. CPT-Drug absorption from the intestinal tract is affected by various factors, including gastrointestinal damage induced by anticancer drugs. In patients with chemotherapy-induced diarrhea, the absorption of lactulose, a marker of paracellular absorption, is increased, whereas the absorption of vitamin A, which requires intracellular processes to be absorbed, is decreased . These fThe aim of this study was to quantitatively evaluate the absorption of digoxin (DGX), a typical probe for P-gp activity, in rats that had been treated with CPT-11. The rats were intravenously administered CPT-11 to induce gastrointestinal damage, and then they were orally administered DGX with or without oral clarithromycin (CAM), a P-gp inhibitor, to determine the pharmacokinetic (PK) parameters of DGX.CPT-11 (7-ethyl-10-[4(− 1-piperidino)-1-piper-idino] carbonyloxy camptothecin; Sawai Pharmaceutical Co., Osaka, Japan), DGX , and CAM  were commercially purchased and used in this study. Unless otherwise noted, all other reagents were commercially purchased from Nacalai Tesque Inc., Tokyo, Japan.Seven-week-old male Sprague-Dawley rats , weighing 200–250 g, were housed under standard conditions for 1 week before the CPT-11 treatment. Throughout the experiments, the rats were housed individually and allowed free access to food and water.Some of the rats were intravenously administered 60 mg/kg of CPT-11 via the tail vein under isoflurane anesthesia once daily for 4 days (the CPT-11 treated group). Three mL/kg saline was administered as a control (the control group). Body weight, food intake, the fecal count, and fecal weight were monitored during the 4-day administration period. On the day after the final dose of CPT-11 was administered (day 5), the state of the rats’ feces and the perianal staining were examined and scored according to the diarrhea score criteria  was administered orally. Ten mg/kg CAM, suspended in 0.5% carboxymethyl cellulose (CMC), was orally administered 5 min before the DGX in the DGX + CAM group. One hundred and fifty-μL blood samples were collected from the jugular vein at 7.5, 15, 30, 60, 120, 180, 360, 720, and 1440 min after the oral administration of DGX. To determine the BA of DGX, 30 μg/kg of DGX  was intravenously injected via the canula on day 6. In the DGX alone and DGX + CAM groups, CMC solution and a CAM suspension, respectively, were orally administered 5 min before the DGX injection. One hundred and fifty-μL blood samples were again collected from the jugular vein at 2, 5, 10, 20, 30, 60, 120, 180, 360, and 720 min after the intravenous administration of DGX. Each blood sample was kept on ice and centrifuged at 3000×t-butyl ether was added, before the sample was vortexed and cooled under 4 °C for 15 min. The samples were centrifuged at 3000×g for 10 min at 4 °C, and 300 μL of the supernatant was evaporated at 50 °C. The residue was dissolved in 100 μL of the mobile phase, and 10 μL of the sample was subjected to LC-MS/MS analysis, as described below.Ten μL of internal standard solution (25 ng/mL digitoxin) was spiked into a glass microtube and then evaporated to dryness, before 50 μL of plasma sample was added. Eight different standard solutions of DGX, with concentrations ranging from 0.03 to 100 ng/mL, were prepared from blank plasma. To each sample, 500 μL of methyl-18-MS-II; Nacalai Tesque, Kyoto, Japan), and a column oven  set at 40 °C. The mobile phase was prepared as an equivalent mixture of aqueous and acetonitrile with 0.1% formic acid, and the flow rate was set at 0.3 mL/min. The MS/MS analysis was conducted in negative-ion mode using electrospray ionization. The DGX and digitoxin ion pairs used for the multiple reaction monitoring in this study were 825.45/779.30 and 809.45/763.25, respectively. The concentrations of the plasma samples were determined using a standard curve obtained via the internal standard method based on the peak area ratio.The DGX concentration was determined using an LC-MS/MS system, consisting of a controller , a pump , a triple quadrupole mass spectrometer , an octadecylsilane column  was defined via log-linear regression during the elimination phase after oral administration. PK parameters were calculated using the following equations:AUC (area under the plasma concentration curve) and AUMC (area under the first moment curve) values were calculated using the trapezoidal method from t = 0 to the final blood sampling point. To assess the plasma concentration of DGX at the time of the intravenous administration of the drug, the y-intercept was determined by extrapolating the line passing through the concentrations at t = 0.033 and 0.083 (hr). The elimination rate constant , [max and BA values of DGX in the control rats by 175 and 45%, respectively.The inhibition extent of CAM in the intestine is determined by the intestinal concentration. In this study, the concentration of CAM in the gastrointestinal tract was considered to exceed 750 μg/mL based on the dosage of CAM (10 mg/kg) and the fluid volume of the rat gastrointestinal tract under fasted conditions (3.2 mL) . Since t8 μg/L),  the dose50, previously reported [Mdr1 knockout rats might be useful.Solubility and permeability are other important factors affecting the intestinal absorption of drugs. In the CPT-11-treated group, the fluid volume in the small intestine might have increased due to the presence of severe diarrhea and reduced the DGX concentration in the intestinal tract, which could have impaired the absorption of the drug. Tanaka et al. reported that an increased luminal fluid volume might impair the absorption of atenolol, a Biopharmaceutics Classification System Class III drug (high solubility and low permeability), but not that of metoprolol  . Since Dreported . Even ifi value of 2.4 μM [DGX is not only a substrate of P-gp, but also a substrate of an intestinal uptake transporter, Oatp1a5 . In thisf 2.4 μM , failed We also made an attempt to carry out everted sac and closed loop intestinal perfusion study in CPT-11-treated rats. However, the intestinal tract was too fragile so that the results showed the intestine became quite leaky or torn. Further study using vesicles prepared from the CPT-11-treated intestine may be required to explain the discrepancy between the protein level and function of P-gp., we have reported that the elimination of DABE was delayed in rats treated with CPT-11, and concluded that impaired bile excretion and/or changes in the distribution volume of DABE induced by CPT-11 treatment might have been responsible because the levels of markers of both renal and liver disorders remained unchanged in the CPT-11-treated rats [iv approximately 10% in both control and CPT-11 treated group. This corresponds to the percentage of bile excretion to the total clearance of DGX in rats, [Although the primary goal of this study was to quantitatively evaluate the change in the gastrointestinal absorption of DGX, the systemic clearance of the drug was also reduced by CPT-11 treatment. Previouslyted rats . Althougin rats,  thus theCPT-11-induced gastrointestinal damage decreased the intestinal absorption of DGX. However, in contrast to the control group, intestinal P-gp inhibition by CAM did not increase the absorption of DGX in the CPT-11 treated group. These results suggest the possibility that P-gp is not responsible for the decreased absorption of DGX, although further studies are necessary to clarify the mechanism.


The Mini-Mental State Examination-Second Edition (MMSE-2) consists of three visions: a brief version (MMSE-2:BV), a standard version (MMSE-2:SV), and an expanded version (MMSE-2: EV). Each version was equipped with alternate forms (blue and red). There was a lack of evidence on the practice effect and test-retest reliability of the three versions of the MMSE-2, limiting its utility in both clinical and research settings. The purpose of this study was to examine the practice effect and test-retest reliability of the MMSE-2 in people with dementia.t-test and Cohen’s d. The test-retest reliability was examined using the intraclass correlation coefficient (ICC).One hundred and twenty participants were enrolled, of which 60 were administered with the blue form twice  and 60 were administered with the blue form first and then the red form . The practice effect was evaluated using a paired p = 0.061–1.000), except for the MMSE-2:SV total score (p = 0.029). In the AF group, no statistically significant differences were found for all three versions of the total scores and subtests (p = 0.106–1.000), except for the visual-constructional ability subtest (p = 0.010). Cohen’s d of all three versions’ total scores and subtests were 0.00–0.20 and 0.00–0.26 for SF group and AF group, respectively. For the test-retest reliability, ICC values for all three versions and eight subtests in SF and AF groups were 0.60–0.93 and 0.56–0.93, respectively.For the practice effects, in the SF group, no statistically significant differences were found for the MMSE-2:BV and MMSE-2: EV total scores and eight subtests . Caution should be taken when interpreting the results of visual-constructional ability, registration, and recall subtests of the MMSE-2.The online version contains supplementary material available at 10.1186/s12877-021-02732-7. It has been estimated that about 46.8 million people are suffering from dementia worldwide and that this figure will increase to 74.7 million by 2030 and to 131.5 million people by 2050 [Four commonly-used cognitive screening tools have been used for the early detection of dementia including the Mini-Mental State Examination (MMSE), the Short Portable Mental Status Questionnaire, the Montreal Cognitive Assessment, and the Saint Louis University Status Examination . Each ofThe MMSE-2 preserves the clinical utility and efficiency of the original MMSE while expanding its application in populations with dementia . The MMSSecondly, each version of the subtests of the MMSE-2 has two alternate forms (blue and red forms) in order to decrease the practice effects that might occur over repeated testing . Almost An assessment would be considered useful if it could produce stable and consistent results with repeated administration . The praA convenience sample of people with dementia was recruited from Department of Psychiatry or Department of Neurology of two teaching hospitals in northern Taiwan between March 2019 and April 2020. The following criteria were used to determine the eligibility of people with dementia to participate in this study: (1) diagnosis of probable dementia and dementia according to the National Institute on Aging and Alzheimer’s Association [Prior to the study, three raters  familiarized themselves with the MMSE-2. The three raters reviewed the user manual of the MMSE-2 and received 4 hours of training from the corresponding author on the administration of the MMSE-2. Then, the three raters performed the MMSE-2 on the corresponding author, and their score results were checked. If there were any discrepancies in the score results, discussions with the corresponding author were required to ensure proper administration procedures and scoring. Finally, the three raters independently administered the MMSE-2 to five people with dementia. The corresponding author observed the assessments and gave MMSE-2 scores simultaneously to confirm that all three raters performed the MMSE-2 correctly in a standardized manner. In addition, when the study began, the raters did not discuss the results of scores with each other to avoid potential bias.The blue form was administered twice in a two-week interval by raters A and B to the participants, who were from hospital A.The alternate forms  were administered by rater C 2 weeks apart to the participants who were from hospital B. The AF group completed the MMSE-2 in a fixed order .All assessments were conducted in a quiet room to avoid interference that might have affected the performance of the participants. Between each assessment, the participants were allowed to break to minimize fatigue. The demographic data of all participants were collected from their medical records.The MMSE-2 was developed to assess cognitive impairment. The MMSE-2: BV (score range: 0–16) is composed of three subtests: registration, orientation, and recall. The MMSE-2: SV (score range: 0–30) is composed of six subtests: three subtests of the MMSE-2:BV along with attention and calculation, language, and visual-constructional ability. The MMSE-2: EV (score range: 0–90) is composed of eight subtests: six subtests of the MMSE-2:SV, as well as story memory and processing speed .Paired  effects . As multrrection  for 11 t2,1) was calculated using a two-way random analysis of variance with absolute agreement. An ICC value of 0.81–1.00 indicates excellent reliability, 0.61–0.80 indicates good reliability, 0.41–0.60 indicates moderate reliability, and < 0.40 indicates poor reliability [95) was calculated on the basis of the standard error of measurement (SEM) to estimate the random measurement error of the MMSE-2 [To examine the test-retest reliability of the MMSE-2, the intraclass correlation coefficient , which was independent of the units of measurement, and used it to determine a relatively true change between two assessments: MDC% =  × 100 . In thist = 1.781, p = 0.080, gender, χ2 = 0.094, p = 0.759, education, t = 39.992, p = 0.105, or CDR, t = 3.436, p = 0.752. The characteristics of the participants are shown in Table A total of 120 participants participated in this study; of these, 60 from hospital A were assigned to the SF group and 60 from hospital B were assigned to the AF group. The participants in the SF group had a mean age of 81.5 ± 7.8 years, 55% were female, and 58.3% had an educational level below elementary school. The participants in the AF group had a mean age of 79.1 ± 6.8 years, 67.3% were female, and 43.3% had an educational level below elementary school. There were no significant group differences in age, p = 0.061–1.000). There was a statistically significant difference in the MMSE-2:SV total score (p = 0.029). The Cohen’s d for all three versions total scores and subtests, except for the visual-constructional ability subtest, ranged from 0.00 to 0.15, indicating no or trivial practice effects. Cohen’s d for the visual-constructional ability subtest was 0.20, indicating a small practice effect , except for the visual-constructional ability subtest (p = 0.010). Cohen’s d for all three versions’ total scores and subtests, except for the visual-constructional ability subtest, ranged from 0.00 to 0.10, indicating no to trivial practice effects. Cohen’s d for the visual-constructional ability subtest was 0.26, indicating a small practice effect , showed good to excellent reliability (ICC = 0.67–0.93) Table . At the In the AF group, all three versions and subtests, except for the registration and recall subtests (ICC = 0.56), showed good to excellent reliability (ICC = 0.67–0.93)  in the AF group showed moderate test-retest reliability, indicating that these two subtests may not consistently assess specific subtest functions over repeated assessments. One possible reason might be that alternate forms (blue and red forms) were used in the AF group, which may have resulted in more variation compared to using the same form as the SF group. Although the alternate forms used diminished the carryover effect, they increased the variation due to random errors in measurement. Thus, caution is needed when using the alternate forms of registration and recall subtests in people with dementia.The MDC values can be used as thresholds to indicate whether an individual’s changed score between two successive assessments is due to real improvement or due to measurement errors . For exaOur results showed that the three versions of the MMSE-2 total scores and all subtests were all below 30% of the highest score of all test data, indicating an acceptable random measurement error. Thus, the MMSE-2 appeared reliable for describing cognitive function in people with dementia. Although the random measurement error was acceptable, we found that the MDC% of the visual construction ability subtest in both groups and the registration subtest in the AF group were higher than those in the other subtests. The large amount of MDC% values indicates that the scores of these two subtests are unstable and thus may obscure the real changes of a person with dementia. Possible reasons for these results might be the nature of practice effects and moderate test-retest reliability. Future studies are needed to investigate the causes of variations in people’s responses to visual-constructional ability and registration subtests between repeated assessments. The results could help users exclude factors that increase random measurement errors and improve the utility of the MMSE-2.p = 0.003), had non-significant differences at baseline (p > 0.05) , the results of the AF group with a sample size of 51were not dramatically different when compared with the previous results using a sample size of 60  used in the AF group might have influenced the results of the study. To confirm our findings, future studies that randomized the alternate form order may be needed. Fourth, the inter-rater reliability between rates has not yet been established, which may jeopardize our current validation of the MMSE-2. Future studies are needed to examine the inter-rater reliability of the MMSE-2 in people with dementia. Fifth, we did not collect information regarding whether the patients had been assessed with the MMSE or MMSE-2 before or during our study, in which case the participants might have become familiar with the screening tools. Familiarity with the screening tools might have caused underestimations of the practice effect and test-retest reliability in this study.Overall, the practice effect of the MMSE-2 diminished when the alternate forms were performed. In addition, the MMSE-2 had good to excellent test-retest reliability with an acceptable random measurement error, which supports the use of this measure in both clinical and research settings. However, the visual-constructional ability subtest showed small practice effects, and both the registration and recall subtests were found to have moderate test-retest reliability. Thus, caution should be exercised when interpreting the results of the visual-constructional ability, registration, and recall subtests of the MMSE-2.Additional file 1. Practice effects of the MMSE-2 in the alternate-form group (AF group).Additional file 2 Results of independent sample t-test of the versions and subtests in the MMSE-2 between the SF (n = 60) and AF (n = 60) groups at baseline.


Streptococcus (S.) mutans and S. sobrinus, produce insoluble and sticky glucans as a biofilm material. The present study demonstrates that a lactic acid bacterium (LAB) named BM53-1 produces a substance that inhibits the sticky glucan synthesis. The BM53-1 strain was isolated from a flower of Actinidia polygama and identified as Lactobacillus reuteri. The substance that inhibits sticky glucan synthesis does not exhibit antibacterial activity against S. mutans. The cariogenic S. mutans produces glucans under the control of three glucosyltransferase (GTF) enzymes, named GtfB, GtfC, and GtfD. Although GtfB and GtfC produce insoluble glucans, GtfD forms soluble glucans. Through quantitative reverse-transcriptional (qRT)-PCR analysis, it was revealed that the BM53-1-derived glucan-production inhibitor (GI) enhances the transcriptions of gtfB and gtfC genes 2- to 7-fold at the early stage of cultivation. However, that of gtfD was not enhanced in the presence of the GI, indicating that the glucan stickiness produced by S. mutans was significantly weaker in the presence of the GI. Our result demonstrates that Lb. reuteri BM53-1 is useful to prevent dental caries.Cariogenic bacteria, such as  Dental caries is caused by local and continuous acid production from carbohydrates by oral bacteria ,2. The bStreptococcus (S.) mutans and S. sobrinus, which are known as cariogenic bacteria, produce insoluble and adhesive glucans from sucrose in the oral cavity  acridine HCl , and benzo[α]pyrene  were dissolved into dimethyl sulfoxide  and used for the assay. One hundred–microliter aliquots of DMSO (negative control), each mutagen (positive control) solution, and the BM53-1 cell suspension were added to 500 μL of a 100 mM phosphate buffer (pH 7.4). If the metabolic activation was evaluated, an S9 mixture  supplemented with cofactor-I  was used instead of the phosphate buffer.Salmonella or E. coli cell suspensions, the assay mixtures were pre-incubated at 37 °C for 20 min with shaking, followed by adding 2 mL of preheated (45 °C) top agar . The assay mixture was immediately poured onto minimal glucose agar plates  and incubated at 37 °C for 48 h. When the number of revertant colonies observed in the assay plate was two times higher than that of the negative control, the result was considered to be mutagenicity-positive. However, if the revertant colony generation had no dose dependence, the results were regarded as mutagenicity-negative.After adding 100 μL of In the assay, BM53-1 cell suspensions were prepared by 2-fold serial dilutions at 5000–312.5 μg/plate, and the concentrations of those mutagens were as follows: AF-2, 0.01 or 0.1 μg/plate; SAZ, 0.5 μg/plate; ICR-191, 1.0 μg/plate; 2-AA, 2.0 or 10.0 μg/plate; and B[α]P, 5.0 μg/plate.S. mutans MT8148R cell was extracted using a NucleoSpin RNA II  according to the manufacturer’s instructions. Using a ReverTra Ace qPCR RT Master Mix with gDNA Remover  enabled the residual DNA fragments to be digested, and the RNA was converted to cDNA according to the manufacturer’s instructions.Total RNA from the gtfB, gtfC, and gtfD were amplified as targets using each cDNA sample as a template with the gene-specific primer sets listed in gyrB and used as a housekeeping gene.The qRT-PCR was performed on the PikoReal Real-Time PCR System  with the KAPA SYBR FAST qPCR Master Mix (2×) Universal . The PCR was performed as follows: an initial 30 s at 95 °C, followed by 40 cycles of 5 s at 95 °C and 30 s at 60 °C. The genes Prior to the molecular mass and constitution sugar analyses, the GI-containing fraction was further purified based on the EPS purification procedure described previously : the conThe molecular mass of the GI was estimated with gel-filtration column  chromatography using a high-performance liquid chromatography (HPLC) system as follows: a 50 mM Tris-HCl buffer (pH 8.0) was used as a mobile phase at a flow rate of 0.5 mL/min. The eluent was divided into 95 fractions every 3 min from 0 to 285 min, and the GI content was monitored using a phenol-sulfuric acid method . The molv/v) acetic acid in methanol and evaporating under the vacuum condition. After three repetitions of the process, the sample was mixed with 0.5 mL of a pyridine/acetic anhydride mixture  and left to stand at 120 °C for 20 min. The reaction mixture was cooled on ice, and the remaining excess amount of reagents was removed by evaporating under the vacuum condition after adding 1.5 mL of water/methanol/toluene . After three repetitions of the process, the residue was mixed with 3 mL of dichloromethane/water , and the dichloromethane fraction was collected. The fraction was dried and finally dissolved into an appropriate volume of acetone.The composition of monosaccharide in the GI was analyzed via the alditol acetate derivatization method  using gam/z range 29–800. The standard monosaccharides were derivatized and analyzed in the same manner as samples. Each peak was confirmed by comparing the retention time and MS with those of the standards, consisting of glucose, xylose, arabinose, mannose, galactose, rhamnose, ribose, and fucose.Each derivative was analyzed with a DB-WAX capillary column (0.25 mm × 0.25 μm × 30 m)  using a JMS-T100GCV AccuTOF GCv 4G gas chromatograph high-resolution time-of-flight mass spectrometer  equipped with a source of ions for electron ionization (EI). GC was performed under the following conditions: split injection mode (50:1), 1 μL injection, injection port temperature 230 °C, and column oven temperature programmed from 50 to 230 °C at 10 °C/min. MS was performed under the following conditions: electron ionization mode , ion source temperature 280 °C, and S. mutans MT8148R or S. sobrinus ATCC27607 as a test organism, and the amount of glucans produced by the test bacterium was compared with that in the absence of LAB. After screening, nine LAB isolates were selected as candidates that have anti-biofilm-forming activity (Actinidia (A.) polygama flower, strongly inhibited the Streptococcus-derived glucan formation. Based on the entire 16S rDNA sequence analysis, the BM53-1 strain was identified as Lb. reuteri.To screen for the LAB strain inhibiting formation of the biofilm material, many LAB strains stored in our library were evaluated using activity , and theS. mutans MT8148R  of cultivation  in the presence of the GI. Although the enhanced expression levels of both genes were maintained after further cultivation (10 h) with the GI, those of gtfB and gtfC decreased 3.2- and 1.6-fold in the absence of the GI. In addition, the gtfD expression level was not significantly affected in the presence of the GI. The comCDE, comS/comR, and sigX expressions, which have been reported to participate in the quorum-sensing signal-transduction system [The qRT-PCR method was used to analyze whether the expression of three GTF genes, f the GI . The expn system ,41,42, dS. mutans, SEM images of the bacterium were captured after cultivation with or without the GI  was about 150 kDa.The averaged molecular mass of the GI purified from the supernatant of BM53-1 cultured in a carrot juice medium, which was estimated based on a gel-filtration HPLC profile, was approximately 30 kDa. On the other hand, that of the water-soluble polysaccharide detected originally in the unfermented carrot juice medium  is remarkably lower than that of the WSPunf .We carried out a kinetic analysis to evaluate the biofilm-inhibitory effect on the GI by calculating the ICunf, only one peak at approximately 25.5 min from the GI is specifically detected and attributed to a hexose, except for the sugars used as standards.The GC–MS profile for the GI indicates that the sample consists of mainly galactose and slight amounts of fucose, arabinose, and mannose . The fewN-acetylglucosamine, N-acetylgalactosamine, and N-acetylmannosamine, was also performed, but attributed peaks were undetectable in all samples , a precursor peptide that is encoded by comS, is secreted to the medium and then incorporated into the bacterial cell to activate the ComR. The Rgg-like regulator ComR positively regulates the sigma-factor encoding gene sigX , which is necessary for the genetic competence of S. mutans. There are some reports on the relationship between XIP and S. mutans showing that (1) the addition of XIP to the medium during cultivation eliminates the biofilm-forming activity and causes a decrease in the expression of gtfB, C, and D; (2) the loss of biofilm depends on the presence of comR; and (3) the expression levels of the three gtf genes are enhanced 10–40 times higher in the comR or comX gene-disrupted mutants than in the parental strain [comS/comR system participates in the changes in gtf expressions observed in the present study; however, the GI fraction did not notably affect the comS/comR-related gene expressions, indicating that changes in the sticky glucan synthesis caused by the BM53-1 strain seem to be due to other mechanisms. How does the GI cause the imbalance of gtf gene expressions? Research to answer this question is now in progress.It is still unclear why the expressions of . mutans . The sigl strain ,53,54. BLb. paracasei IJH-SONE68 has been reported to display prevention effect in picryl chloride-induced contact dermatitis model mice [http://www.umin.ac.jp/ctr/index.htm, accessed on 16 May 2021). Therefore, although the GI is predicted to be a relatively short polysaccharide with an average molecular weight of 30 kDa, the GI may have an anti-inflammatory effect as other EPSs we have previously reported.Our recent reports have shown that some EPSs produced from the plant-derived LAB strains have a hyaluronidase-inhibitory effect ,56,57, wdel mice . FurtherS. mutans-infected model mice. The clinical trial will also be planned in the near future for the evaluation of preventive effect for dental caries and periodontal inflammation.For an industrial application of the GI to develop the oral health-care products, there are still some issues that we have to settle. Human saliva consists of mostly water and other secreted proteins, such as enzymes and immunoglobulins, to aid digestion of foods and protection from infectious disease . As descS. mutans is regarded as the major pathogen of the disease [Lactobacillus reuteri BM53-1 produces a substance that causes the stickiness of glucan to disappear, through unbalance of the expression levels of three gtf genes of S. mutans.Although many kinds of bacteria are involved in the occurrence and development of dental caries,  disease ,62,63. L